The pedunculopontine (PPTg) and laterodorsal tegmental nuclei (LDTg) provide cholinergic, GABAergic and glutamatergic afferents to the ventral tegmental area (VTA). We recently demonstrated that the PPTg and the LDTg contain independent populations of cholinergic, glutamatergic and GABAergic neurons (Wang &Morales, EJN, 2009). Here, by combining retrograde tract tracing and in situ hybridization, we investigated the proportion of PPTg/LDTg cholinergic, glutamatergic and GABAergic neurons projecting to the VTA. The retrograde tract tracer Fluoro-Gold (FG) was delivered into the VTA and detected by immunohistochemistry. The neuronal phenotype of retrogradely labeled FG neurons was established by investigating cellular co-expression of FG and transcripts encoding choline acetyltransferase (ChAT;cholinergic marker), glutamic acid decarboxylase (GAD;GABAergic marker) or the vesicular glutamate transporter 2 (vGluT2;glutamatergic marker). Within the PPTg, only 20% (rostral) to 12% (caudal) of the FG neurons co-expressed ChAT mRNA, and 30% (rostral) to 6% (caudal) co-expressed GAD mRNA. In contrast, the majority of FG neurons co-expressed vGluT2 mRNA from the rostral (53%) to caudal (82%) levels. These data indicate that the major input from the PPTg to the VTA is glutamatergic rather than cholinergic. With regard to the LDTg, the FG neurons expressing ChAT mRNA showed a gradual rostro-caudal increase (13% to 28%, respectively), as opposed to the gradual rostro-caudal decrease in FG neurons expressing GAD mRNA (62% to 38%, rostro-caudal distribution). In contrast to the uneven rostro-caudal distribution of both FG/ChAT and FG/GAD neurons in the LDTg, the rostro-caudal distribution of FG neurons expressing vGluT2 mRNA was relatively uniform throughout the LDTg (44% to 50%, rostro-caudal distribution). These data indicated that the major input from the LDTg to the VTA is not cholinergic, but rather glutamatergic or GABAergic. In summary, our results showed that (1) glutamatergic, cholinergic and GABAergic neurons from both the PPTg and the LDTg differentially innervate the VTA, (2) the major input from the PPTg to the VTA is from glutamatergic neurons, and not from cholinergic neurons, (3) the major input from the LDTg to the VTA is not from cholinergic neurons, but from either glutamatergic or GABAergic neurons. In conclusion, despite the fact that glutamatergic, cholinergic and GABAergic neurons from both the PPTg and the LDTg innervate the VTA, these two nuclei are likely to have differential effects on VTA neurotransmission, as the proportion in which their cholinergic, glutamatergic and GABAergic afferents target the VTA is distinct.